Treatment with anti-vegf antibodies

ABSTRACT

This invention concerns in general treatment of diseases and pathological conditions with anti-VEGF antibodies. More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer using an anti-VEGF antibody, preferably in combination with one or more additional anti-tumor therapeutic agents.

This is a continuation application claiming priority to U.S. applicationSer. No. 13/602,619, filed Sep. 4, 2012, which is a continuationapplication claiming priority to U.S. application Ser. No. 13/355,205,filed Jan. 20, 2012, which is a continuation application claimingpriority to U.S. application Ser. No. 13/019,414, filed Feb. 2, 2011,which is a continuation application claiming priority to U.S.application Ser. No. 12/576,085, filed Oct. 8, 2009, which is acontinuation application claiming priority to U.S. application Ser. No.11/763,263, filed Jun. 14, 2007, which is a continuation application ofU.S. application Ser. No. 10/857,249, filed May 28, 2004, which is anon-provisional application claiming priority to U.S. provisionalApplication No. 60/474,480, filed May 30, 2003, the contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates in general to treatment of human diseases andpathological conditions. More specifically, the invention relates toanti-angiogenesis therapy of cancer, either alone or in combination withother anti-cancer therapies.

BACKGROUND OF THE INVENTION

Cancer remains to be one of the most deadly threats to human health. Inthe U.S., cancer affects nearly 1.3 million new patients each year, andis the second leading cause of death after heart disease, accounting forapproximately 1 in 4 deaths. It is also predicted that cancer maysurpass cardiovascular diseases as the number one cause of death within5 years. Solid tumors are responsible for most of those deaths. Althoughthere have been significant advances in the medical treatment of certaincancers, the overall 5-year survival rate for all cancers has improved,only by about 10% in the past 20 years. Cancers, or malignant tumors,metastasize and grow rapidly in an uncontrolled manner, making timelydetection and treatment extremely difficult. Furthermore, cancers canarise from almost any tissue in the body through malignanttransformation of one or a few normal cells within the tissue, and eachtype of cancer with particular tissue origin differs from the others.

Current methods of cancer treatment are relatively non-selective.Surgery removes the diseased tissue; radiotherapy shrinks solid tumors;and chemotherapy kills rapidly dividing cells. Chemotherapy, inparticular, results in numerous side effects, in some cases so severe asto limit the dosage that can be given and thus preclude the use ofpotentially effective drugs. Moreover, cancers often develop resistanceto chemotherapeutic drugs.

Thus, there is an urgent need for specific and more effective cancertherapies.

Angiogenesis is an important cellular event in which vascularendothelial cells proliferate, prune and reorganize to form new vesselsfrom preexisting vascular network. There are compelling evidences thatthe development of a vascular supply is essential for normal andpathological proliferative processes (Folkman and Klagsbrun (1987)Science235:442-447). Delivery of oxygen and nutrients, as well as theremoval of catabolic products, represent rate-limiting steps in themajority of growth processes occurring in multicellular organisms. Thus,it has been generally assumed that the vascular compartment isnecessary, not only for organ development and differentiation duringembryogenesis, but also for wound healing and reproductive functions inthe adult.

Angiogenesis is also implicated in the pathogenesis of a variety ofdisorders, including but not limited to, tumors, proliferativeretinopathies, age-related macular degeneration, rheumatoid arthritis(RA), and psoriasis. Angiogenesis is essential for the growth of mostprimary tumors and their subsequent metastasis. Tumors can absorbsufficient nutrients and oxygen by simple diffusion up to a size of 1-2mm, at which point their further growth requires the elaboration ofvascular supply. This process is thought to involve recruitment of theneighboring host mature vasculature to begin sprouting new blood vesselcapillaries, which grow towards, and subsequently infiltrate, the tumormass. In addition, tumor angiogenesis involve the recruitment ofcirculating endothelial precursor cells from the bone marrow to promoteneovascularization. Kerbel (2000) Carcinogenesis 21:505-515; Lynden etal. (2001) Nat. Med. 7:1194-1201.

While induction of new blood vessels is considered to be the predominantmode of tumor angiogenesis, recent data have indicated that some tumorsmay grow by co-opting existing host blood vessels. The co-optedvasculature then regresses, leading to tumor regression that iseventually reversed by hypoxia-induced angiogenesis at the tumor margin.Holash et al. (1999) Science 284:1994-1998.

In view of the remarkable physiological and pathological importance ofangiogenesis, much work has been dedicated to the elucidation of thefactors capable of regulating this process. It is suggested that theangiogenesis process is regulated by a balance between pro- andanti-angiogenic molecules, and is derailed in various diseases,especially cancer. Carmeliet and Jain (2000) Nature 407:249-257.

Vascular endothelial cell growth factor (VEGF), which is also termedVEGF-A or vascular permeability factor (VPF), has been reported as apivotal regulator of both normal and abnormal angiogenesis. Ferrara andDavis-Smyth (1997) Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med.77:527-543. Compared to other growth factors that contribute to theprocesses of vascular formation, VEGF is unique in its high specificityfor endothelial cells within the vascular system. VEGF is essential forembryonic vasculogenesis and angiogenesis. Carmeliet et al. (1996)Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442.Furthermore, VEGF is required for the cyclical blood vesselproliferation in the female reproductive tract and for bone growth andcartilage formation. Ferrara et al. (1998) Nature Med. 4:336-340; Gerberet al. (1999) Nature Med. 5:623-628.

In addition to being an angiogenic factor in angiogenesis andvasculogenesis, VEGF, as a pleiotropic growth factor, exhibits multiplebiological effects in other physiological processes, such as endothelialcell survival, vessel permeability and vasodilation, monocyte chemotaxisand calcium influx. Ferrara and Davis-Smyth (1997), supra. Moreover,recent studies have reported mitogenic effects of VEGF on a fewnon-endothelial cell types, such as retinal pigment epithelial cells,pancreatic duct cells and Schwann cells. Guerrin et al. (1995) J. CellPhysiol. 164:385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol.126:125-132; Sondell et al. (1999) J. Neurosci. 19:5731-5740.

Substantial evidence also implicates VEGF's critical role in thedevelopment of conditions or diseases that involve pathologicalangiogenesis. The VEGF mRNA is overexpressed by the majority of humantumors examined (Berkman et al. J Clin Invest 91:153-159 (1993); Brownet al. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res.53:4727-4735 (1993); Mattern et al. Brit. J. Cancer. 73:931-934 (1996);and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, theconcentration of VEGF in eye fluids are highly correlated to thepresence of active proliferation of blood vessels in patients withdiabetic and other ischemia-related retinopathies (Aiello et al. N.Engl. J. Med. 331:1480-1487 (1994)). Furthermore, recent studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci.37:855-868 (1996)).

Given its central role in promoting tumor growth, VEGF provides anattractive target for therapeutic intervention. Indeed, a variety oftherapeutic strategies aimed at blocking VEGF or its receptor signalingsystem are currently being developed for the treatment of neoplasticdiseases. Rosen (2000) Oncologist 5:20-27; Ellis et al. (2000)Oncologist 5:11-15; Kerbel (2001) J. Clin. Oncol. 19:45S-51S. So far,VEGF/VEGF receptor blockade by monoclonal antibodies and inhibition ofreceptor signaling by tyrosine kinase inhibitors are the best studiedapproaches. VEGFR-1 ribozymes, VEGF toxin conjugates, and soluble VEGFreceptors are also being investigated.

The anti-VEGF antibody “Bevacizumab (BV)”, also known as “rhuMAb VEGF”or “Avastin™”, is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of Bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated. Bevacizumab is being investigated clinically for treatingvarious cancers, and some early stage trials have shown promisingresults. Kerbel (2001) J. Clin. Oncol. 19:45S-51S; De Vore et al. (2000)Proc. Am. Soc. Clin. Oncol. 19:485a; Johnson et al. (2001) Proc. Am.Soc. Clin. Oncol. 20:315a; Kabbinavar et al. (2003)J. Clin. Oncol.21:60-65.

SUMMARY OF THE INVENTION

The present invention concerns methods of using anti-VEGF antibody fortreating diseases and pathological conditions. In particular, theinvention provides an effective approach for treating cancers, partiallybased on the unexpected results that adding anti-VEGF antibody to astandard chemotherapy results in statistically significant andclinically meaningful improvements among cancer patients.

Accordingly, in one aspect, the invention provides a method of treatingcancer in a human patient, comprising administering to the patienteffective amounts of an anti-VEGF antibody and an anti-neoplasticcomposition, wherein said anti-neoplastic composition comprises at leastone chemotherapeutic agent.

The cancer amendable for treatment by the present invention include, butnot limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includesquamous cell cancer, lung cancer (including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung), cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer (including gastrointestinal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. Preferably, thecancer is selected from the group consisting of breast cancer,colorectal cancer, rectal cancer, non-small cell lung cancer,non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, livercancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. More preferably, the cancer iscolorectal cancer. The cancerous conditions amendible for treatment ofthe invention include metastatic cancers. The method of the presentinvention is particularly suitable for the treatment of vascularizedtumors.

Any chemotherapeutic agent exhibiting anticancer activity can be usedaccording to the present invention. Preferably, the chemotherapeuticagent is selected from the group consisting of alkylating agents,antimetabolites, folic acid analogs, pyrimidine analogs, purine analogsand related inhibitors, vinca alkaloids, epipodopyyllotoxins,antibiotics, L-Asparaginase, topoisomerase inhibitor, interferons,platinum coordination complexes, anthracenedione substituted urea,methyl hydrazine derivatives, adrenocortical suppressant,adrenocorticosteroides, progestins, estrogens, anti estrogen, androgens,anti androgen, and gonadotropin-releasing hormone analog. Morepreferably, the chemotherapeutic agent is selected from the groupconsisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan,oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or morechemotherapeutic agents can be used in a cocktail to be administered incombination with administration of the anti-VEGF antibody. One preferredcombination chemotherapy is fluorouracil-based, comprising 5-FU and oneor more other chemotherapeutic agent(s). Suitable dosing regimens ofcombination chemotherapies are known in the art and described in, forexample, Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al.(2000) Lancet 355: 1041-7.

In one aspect, the present invention provides a method for increasingthe duration of survival of a human patient having cancer, comprisingadministering to the patient effective amounts of an anti-VEGF antibodycomposition and an anti-neoplastic composition, wherein saidanti-neoplastic composition comprises at least one chemotherapeuticagent, whereby the co-administration of the anti-VEGF antibody and theanti-neoplastic composition effectively increases the duration ofsurvival.

In another aspect, the present invention provides a method forincreasing the progression free survival of a human patient havingcancer, comprising administering to the patient effective amounts of ananti-VEGF antibody composition and an anti-neoplastic composition,wherein said anti-neoplastic composition comprises at least onechemotherapeutic agent, whereby the co-administration of the anti-VEGFantibody and the anti-neoplastic composition effectively increases theduration of progression free survival.

Furthermore, the present invention provides a method for treating agroup of human patients having cancer, comprising administering to thepatient effective amounts of an anti-VEGF antibody composition and ananti-neoplastic composition, wherein said anti-neoplastic compositioncomprises at least one chemotherapeutic agent, whereby theco-administration of the anti-VEGF antibody and the anti-neoplasticcomposition effectively increases the response rate in the group ofpatients.

In yet another aspect, the present invention provides a method forincreasing the duration of response of a human patient having cancer,comprising administering to the patient effective amounts of ananti-VEGF antibody composition and an anti-neoplastic composition,wherein said anti-neoplastic composition comprises at least onechemotherapeutic agent, whereby the co-administration of the anti-VEGFantibody and the anti-neoplastic composition effectively increases theduration of response.

The invention also provides a method of treating a human patientsusceptible to or diagnosed with colorectal cancer, comprisingadministering to the patient effective amounts of an anti-VEGF antibody.The colorectal cancer can be metastatic. The anti-VEGF antibodytreatment can be further combined with a standard chemotherapy forcolorectal cancer such as the Saltz (5-FU/LV/irinotecan) regimendescribed by Saltz et al. (1999).

In one preferred embodiment, the invention provides a method of treatinga human patient or a group of human patients having metastaticcolorectal cancer, comprising administering to the patient effectiveamounts of an anti-VEGF antibody composition and an anti-neoplasticcomposition, wherein said anti-neoplastic composition comprises at leastone chemotherapeutic agent, whereby the co-administration of theanti-VEGF antibody and the anti-neoplastic composition results instatistically significant and clinically meaningful improvement of thetreated patient as measured by the duration of survival, progressionfree survival, response rate or duration of response. Preferably, theanti-neoplastic composition is a fluorouracil based combination regimen.More preferably the combination regimen comprises 5-FU+leucovorin,5-FU+leucovorin+irinotecan (IFL), or 5-FU+leucorvin+oxaliplatin(FOLFOX).

The invention provides an article of manufacture comprising a container,a composition within the container comprising an anti-VEGF antibody anda package insert instructing the user of the composition to administerto a cancer patient the anti-VEGF antibody composition and ananti-neoplastic composition comprising at least one chemotherapeuticagent.

The invention also provides a kit for treating cancer in a patientcomprising a package comprising an anti-VEGF antibody composition andinstructions for using the anti-VEGF antibody composition and ananti-neoplastic composition comprising at least one chemotherapeuticagent for treating cancer in a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents Kaplan-Meier estimates of survival. The medianduration of survival (indicated by the dotted lines) was 20.3 months inthe group given irinotecan, fluorouracil, and leucovorin (IFL) plusbevacizumab, as compared with 15.6 months in the group given TFL plusplacebo, corresponding to a hazard ratio for death of 0.66 (P<0.001).

FIG. 2 represents Kaplan-Meier estimates of progression-free survival.The median duration of progression-free survival (indicated by thedotted lines) was 10.6 months in the group given irinotecan,fluorouracil, and leucovorin (IFL) plus bevacizumab, as compared with6.2 months in the group given IFL plus placebo, corresponding to ahazard ratio for progression of 0.54 (P<0.001).

FIGS. 3A-3C provide analysis of duration of survival by differentsubgroups of patients divided by baseline characteristics.

FIG. 4 represents Kaplan-Meier estimates of survival comparing the groupgiven 5-FU/LV plus placebo vs. the group given 5-FU/LV plus bevacizumab(BV).

FIG. 5 represents Kaplan-Meier estimates of progression-free survivalcomparing the group given 5-FU/LV plus placebo vs. the group given5-FU/LV plus bevacizumab (BY).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “VEGF” and “VEGF-A” are used interchangeably to refer to the165-amino acid vascular endothelial cell growth factor and related 121-,189-, and 206-amino acid vascular endothelial cell growth factors, asdescribed by Leung et al. Science, 246:1306 (1989), and Houck et al.Mol. Endocrin., 5:1806 (1991), together with the naturally occurringallelic and processed forms thereof. The term “VEGF” is also used torefer to truncated forms of the polypeptide comprising amino acids 8 to109 or 1 to 109 of the 165-amino acid human vascular endothelial cellgrowth factor. Reference to any such forms of VEGF may be identified inthe present application, e.g., by “VEGF (8-109),” “VEGF (1-109)” or“VEGF₁₆₅.” The amino acid positions for a “truncated” native VEGF arenumbered as indicated in the native VEGF sequence. For example, aminoacid position 17 (methionine) in truncated native VEGF is also position17 (methionine) in native VEGF. The truncated native VEGF has bindingaffinity for the KDR and Flt-1 receptors comparable to native VEGF.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. Preferably, the anti-VEGF antibodyof the invention can be used as a therapeutic agent in targeting andinterfering with diseases or conditions wherein the VEGF activity isinvolved. An anti-VEGF antibody will usually not bind to other VEGFhomologues such as VEGF-B or VEGF-C, nor other growth factors such asP1GF, PDGF or bFGF. A preferred anti-VEGF antibody is a monoclonalantibody that binds to the same epitope as the monoclonal anti-VEGFantibody A4.6.1 produced by hybridoma ATCC HB 10709. More preferably theanti-VEGF antibody is a recombinant humanized anti-VEGF monoclonalantibody generated according to Presta et al. (1997) Cancer Res.57:4593-4599, including but not limited to the antibody known asbevacizumab (BV; Avastin™).

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including its binding to one or more VEGF receptors. VEGFantagonists include anti-VEGF antibodies and antigen-binding fragmentsthereof, receptor molecules and derivatives which bind specifically toVEGF thereby sequestering its binding to one or more receptors,anti-VEGF receptor antibodies and VEGF receptor antagonists such assmall molecule inhibitors of the VEGFR tyrosine kinases.

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), expressly incorporated herein by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG1 EU antibody.

A “native sequence” polypeptide comprises a polypeptide having the sameamino acid sequence as a polypeptide derived from nature. Thus, a nativesequence polypeptide can have the amino acid sequence ofnaturally-occurring polypeptide from any mammal. Such native sequencepolypeptide can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence” polypeptidespecifically encompasses naturally-occurring truncated or secreted formsof the polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide. Such variants include, for instance, polypeptides whereinone or more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. Ordinarily, a variant will have at leastabout 80% amino acid sequence identity, more preferably at least about90% amino acid sequence identity, and even more preferably at leastabout 95% amino acid sequence identity with the native sequencepolypeptide.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments (see below) so long as they exhibit the desired biologicalactivity.

Unless indicated otherwise, the expression “multivalent antibody” isused throughout this specification to denote an antibody comprisingthree or more antigen binding sites. The multivalent antibody ispreferably engineered to have the three or more antigen binding sitesand is generally not a native sequence IgM or IgA antibody.

“Antibody fragments” comprise only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the present definition include: (i) the Fabfragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1domains; (iv) the Fd′ fragment having VH and CH1 domains and one or morecysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) whichconsists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)₂fragments, a bivalent fragment including two Fab′ fragments linked by adisulphide bridge at the hinge region; (ix) single chain antibodymolecules (e.g. single chain Fv; scFv) (Bird et al., Science 242:423-426(1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x)“diabodics” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi)“linear antibodies” comprising a pair of tandem Fd segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” is not to be construed as requiring production ofthe antibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) or Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art. In one embodiment, the human antibody is selected froma phage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al.PNAS (USA) 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol.,227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Humanantibodies can also be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the humanantibody may be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Preferred affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

A “functional antigen binding site” of an antibody is one which iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same. For the multimericantibodies herein, the number of functional antigen binding sites can beevaluated using ultracentrifugation analysis as described in Example 2below. According to this method of analysis, different ratios of targetantigen to multimeric antibody are combined and the average molecularweight of the complexes is calculated assuming differing numbers offunctional binding sites. These theoretical values are compared to theactual experimental values obtained in order to evaluate the number offunctional binding sites.

An antibody having a “biological characteristic” of a designatedantibody is one which possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

An “agonist antibody” is an antibody which binds to and activates areceptor. Generally, the receptor activation capability of the agonistantibody will be at least qualitatively similar (and may be essentiallyquantitatively similar) to a native agonist ligand of the receptor. Anexample of an agonist antibody is one which binds to a receptor in theTNF receptor superfamily and induces apoptosis of cells expressing theTNF receptor. Assays for determining induction of apoptosis aredescribed in WO98/51793 and WO99/37684, both of which are expresslyincorporated herein by reference.

A “disorder” is any condition that would benefit from treatment with theantibody. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include benign and malignant tumors; leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the durationof survival, time to disease progression (TTP), the response rates (RR),duration of response, and/or quality of life.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

The term “mammalian host” as used herein refers to any compatibletransplant recipient. By “compatible” is meant a mammalian host thatwill accept the donated graft. Preferably, the host is human. If boththe donor of the graft and the host are human, they are preferablymatched for HLA class II antigens so as to improve histocompatibility.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent capableof inhibiting or preventing tumor growth or function, and/or causingdestruction of tumor cells. Therapeutic agents suitable in ananti-neoplastic composition for treating cancer include, but not limitedto, chemotherapeutic agents, radioactive isotopes, toxins, cytokinessuch as interferons, and antagonistic agents targeting cytokines,cytokine receptors or antigens associated with tumor cells. For example,therapeutic agents useful in the present invention can be antibodiessuch as anti-HER2 antibody and anti-CD20 antibody, or small moleculetyrosine kinase inhibitors such as VEGF receptor inhibitors and EGFreceptor inhibitors. Preferably the therapeutic agent is achemotherapeutic agent.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem Intl. Ed. Engl. 33:183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-di azo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum coordination complexes such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone;teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON. toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell in vitro and/or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL®, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); epidermal growth factor; hepatic growthfactor; fibroblast growth factor; prolactin; placental lactogen; tumornecrosis factor-alpha and -beta; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-alpha; platelet-growth factor; transforming growth factors (TGFs)such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, -beta and -gamma colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocytemacrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; atumor necrosis factor such as TNF-alpha or TNF-beta; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

The term “intravenous infusion” refers to introduction of a drug intothe vein of an animal or human patient over a period of time greaterthan approximately 5 minutes, preferably between approximately 30 to 90minutes, although, according to the invention, intravenous infusion isalternatively administered for 10 hours or less.

The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, preferably 5minutes or less.

The term “subcutaneous administration” refers to introduction of a drugunder the skin of an animal or human patient, preferable within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle. The pocket may be created by pinchingor drawing the skin up and away from underlying tissue.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery ispreferably less than approximately 15 minutes, more preferably less than5 minutes, and most preferably less than 60 seconds. Administration ispreferably within a pocket between the skin and underlying tissue, wherethe pocket is created, for example,—by pinching or drawing the skin upand away from underlying tissue.

An “angiogenic factor” is a growth factor which stimulates thedevelopment of blood vessels. The preferred angiogenic factor herein isVascular Endothelial Growth Factor (VEGF).

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

II. Production of Anti-VEGF Antibodies A. Antibody Preparation (i) VEGFAntigen

Means for preparing and characterizing antibodies are well known in theart. A description follows as to exemplary techniques for the productionof anti-VEGF antibodies used in accordance with the present invention.The VEGF antigen to be used for production of antibodies may be, e.g.,the VEGF₁₆₅ molecule as well as other isoforms of VEGF or a fragmentthereof containing the desired epitope. Other forms of VEGF useful forgenerating anti-VEGF antibodies of the invention will be apparent tothose skilled in the art.

Human VEGF was obtained by first screening a cDNA library prepared fromhuman cells, using bovine VEGF cDNA as a hybridization probe. Leung etal. (1989) Science, 246:1306. One cDNA identified thereby encodes a165-amino acid protein having greater than 95% homology to bovine VEGF;this 165-amino acid protein is typically referred to as human VEGF(hVEGF) or VEGF₁₆₅. The mitogenic activity of human VEGF was confirmedby expressing the human VEGF cDNA in mammalian host cells. Mediaconditioned by cells transfected with the human VEGF cDNA promoted theproliferation of capillary endothelial cells, whereas control cells didnot. Leung et al. (1989) Science, supra.

Although a vascular endothelial cell growth factor could be isolated andpurified from natural sources for subsequent therapeutic use, therelatively low concentrations of the protein in follicular cells and thehigh cost, both in terms of effort and expense, of recovering VEGFproved commercially unavailing. Accordingly, further efforts wereundertaken to clone and express VEGF via recombinant DNA techniques.(See, e.g., Ferrara (1995) Laboratory Investigation 72:615-618, and thereferences cited therein).

VEGF is expressed in a variety of tissues as multiple homodimeric forms(121, 145, 165, 189, and 206 amino acids per monomer) resulting fromalternative RNA splicing. VEGF₁₂₁ is a soluble mitogen that does notbind heparin; the longer forms of VEGF bind heparin with progressivelyhigher affinity. The heparin-binding forms of VEGF can be cleaved in thecarboxy terminus by plasmin to release a diffusible form(s) of VEGF.Amino acid sequencing of the carboxy terminal peptide identified afterplasmin cleavage is Arg₁₁₀-Ala₁₁₁. Amino terminal “core” protein, VEGF(1-110) isolated as a homodimer, binds neutralizing monoclonalantibodies (such as the antibodies referred to as 4.6.1 and 3.2E3.1.1)and soluble forms of VEGF receptors with similar affinity compared tothe intact VEGF₁₆₅ homodimer.

Several molecules structurally related to VEGF have also been identifiedrecently, including placenta growth factor (PIGF), VEGF-B, VEGF-C,VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev., supra;Ogawa et al. (1998) J. Biological Chem. 273:31273-31281; Meyer et al.(1999) EMBO J., 18:363-374. A receptor tyrosine kinase, Flt-4 (VEGFR-3),has been identified as the receptor for VEGF-C and VEGF-D. Joukov et al.(1996) EMBO. J. 15:1751; Lee et al. (1996) Proc. Natl. Acad. Sci. USA93:1988-1992; Achen et al. (1998) Proc. Natl. Acad. Sci. USA 95:548-553.VEGF-C has recently been shown to be involved in the regulation oflymphatic angiogenesis. Jeltsch et al. (1997) Science 276:1423-1425.

Two VEGF receptors have been identified, Flt-1 (also called VEGFR-1) andKDR (also called VEGFR-2). Shibuya et al. (1990) Oncogene 8:519-527; deVries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem.Biophys. Res. Commun. 187:1579-1586. Neuropilin-1 has been shown to be aselective VEGF receptor, able to bind the heparin-binding VEGF isoforms(Soker et al. (1998) Cell 92:735-45). Both Flt-1 and KDR belong to thefamily of receptor tyrosine kinases (RTKs). The RTKs comprise a largefamily of transmembrane receptors with diverse biological activities. Atpresent, at least nineteen (19) distinct RTK subfamilies have beenidentified. The receptor tyrosine kinase (RTK) family includes receptorsthat are crucial for the growth and differentiation of a variety of celltypes (Yarden and Ullrich (1988) Ann. Rev. Biochem. 57:433-478; Ullrichand Schlessinger (1990) Cell 61:243-254). The intrinsic function of RTKsis activated upon ligand binding, which results in phosphorylation ofthe receptor and multiple cellular substrates, and subsequently in avariety of cellular responses (Ullrich & Schlessinger (1990) Cell61:203-212). Thus, receptor tyrosine kinase mediated signal transductionis initiated by extracellular interaction with a specific growth factor(ligand), typically followed by receptor dimerization, stimulation ofthe intrinsic protein tyrosine kinase activity and receptortrans-phosphorylation. Binding sites are thereby created forintracellular signal transduction molecules and lead to the formation ofcomplexes with a spectrum of cytoplasmic signaling molecules thatfacilitate the appropriate cellular response. (e.g., cell division,differentiation, metabolic effects, changes in the extracellularmicroenvironment) see, Schlessinger and Ullrich (1992) Neuron 9:1-20.Structurally, both Flt-1 and KDR have seven immunoglobulin-like domainsin the extracellular domain, a single transmembrane region, and aconsensus tyrosine kinase sequence which is interrupted by akinase-insert domain. Matthews et al. (1991) Proc. Natl. Acad. Sci. USA88:9026-9030; Terman et al. (1991) Oncogene 6:1677-1683.

(ii) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(iii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iv) Humanized and human antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)).

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Examples of BsAbs include those with onearm directed against a tumor cell antigen and the other arm directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell ahesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α(IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcγRI, FcγRII or FcγRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/antiherpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs for tumordetection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants; and BsAbs as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase. Examples of trispecific antibodiesinclude anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986). According to anotherapproach described in WO96/27011, the interface between a pair ofantibody molecules can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the C₁₁3 domain of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the VEGF receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vii) Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

(viii) Immunoconjugates

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alphasarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugate antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bisazido compounds (such asbis-(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

(ix) Immunoliposomes

The antibody disclosed herein may also be formulated as immunoliposomes.

Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980);and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484 (1989)

(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugatc useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to convertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosinc into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibody bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984)).

(xi) Antibody-Salvage Receptor Binding Epitope Fusions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g. by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g., by DNA or peptide synthesis).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

(xii) Other Covalent Modifications of the Antibody

Covalent modifications of the antibody are included within the scope ofthis invention. They may be made by chemical synthesis or by enzymaticor chemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, pchloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al. Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onantibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350(1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

B. Vectors, Host Cells and Recombinant Methods

The anti-VEGF antibody of the invention can be produced recombinantly,using techniques and materials readily obtainable.

For recombinant production of an anti-VEGF antibody, the nucleic acidencoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the monoclonal antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophcnolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,omithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoA promoter, β-lactamase and lactose promoter systems, alkalinephosphatasc, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv (1982) Nature 297:17-18 on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe anti-VEGF antibody and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

III. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibodyformulations are described in WO 97/04801, expressly incorporated hereinbe reference.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to EGFR, VEGF (e.g. an antibody which binds a different epitope onVEGF), VEGFR, or ErbB2 (e.g., Herceptin®) in the one formulation.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine, growth inhibitory agent and/or small molecule VEGFRantagonist. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

IV. Therapeutic Uses of Anti-VEGF Antibodies

It is contemplated that, according to the present invention, theanti-VEGF antibodies may be used to treat various neoplasms ornon-neoplastic conditions characterized by pathological angiogenesis.Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, chronic inflammation, lunginflammation, nephrotic syndrome, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), and pleuraleffusion.

The antibodies of the invention are preferably used in the treatment oftumors in which angiogenesis plays an important role in tumor growth,including cancers and benign tumors. Examples of cancer to be treatedherein include, but are not limited to, carcinoma, lymphoma, blastoma,sarcoma, and leukemia. More particular examples of such cancers includesquamous cell cancer, lung cancer (including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung), cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer (including gastrointestinal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. More particularly,cancers that are amenable to treatment by the antibodies of theinvention include breast cancer, colorectal cancer, rectal cancer,non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cellcancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissuesarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,melanoma, ovarian cancer, mesothelioma, and multiple myeloma. Morepreferably, the methods of the invention are used to treat colorectalcancer in a human patient.

The present invention encompasses antiangiogenic therapy, a novel cancertreatment strategy aimed at inhibiting the development of tumor bloodvessels required for providing nutrients to support tumor growth.Because angiogenesis is involved in both primary tumor growth andmetastasis, the antiangiogenic treatment provided by the invention iscapable of inhibiting the neoplastic growth of tumor at the primary siteas well as preventing metastasis of tumors at the secondary sites,therefore allowing attack of the tumors by other therapeutics.

Combination Therapies

It is contemplated that when used to treat various diseases such astumors, the antibodies of the invention can be combined with othertherapeutic agents suitable for the same or similar diseases. When usedfor treating cancer, antibodies of the present invention may be used incombination with conventional cancer therapies, such as surgery,radiotherapy, chemotherapy or combinations thereof.

In certain aspects, other therapeutic agents useful for combinationcancer therapy with the antibody of the invention include otheranti-angiogenic agents. Many anti-angiogenic agents have been identifiedand are known in the arts, including those listed by Carmeliet and Jain(2000). Preferably, the anti-VEGF antibody of the invention is used incombination with another VEGF antagonist or a VEGF receptor antagonistsuch as VEGF variants, soluble VEGF receptor fragments, aptamers capableof blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, lowmolecule weight inhibitors of VEGFR tyrosine kinases and anycombinations thereof. Alternatively, or in addition, two or moreanti-VEGF antibodies may be co-administered to the patient.

In some other aspects, other therapeutic agents useful for combinationtumor therapy with the antibody of the invention include antagonist ofother factors that are involved in tumor growth, such as EGFR, ErbB2(also known as Her2) ErbB3, ErbB4, or TNF. Sometimes, it may bebeneficial to also administer one or more cytokines to the patient. In apreferred embodiment, the VEGF antibody is co-administered with a growthinhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by the VEGF antibody. However, simultaneousadministration or administration of the VEGF antibody first is alsocontemplated. Suitable dosages for the growth inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth inhibitory agent and anti-VEGF antibody.

Chemotherapeutic Agents

In certain aspects, the present invention provides a method of treatingcancer, by administering effective amounts of an anti-VEGF antibody andone or more chemotherapeutic agents to a patient susceptible to, ordiagnosed with, cancer. A variety of chemotherapeutic agents may be usedin the combined treatment methods of the invention. An exemplary andnon-limiting list of chemotherapeutic agents contemplated is providedherein under “Definition”.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. Variation in dosage will likely occur depending onthe condition being treated. The physician administering treatment willbe able to determine the appropriate dose for the individual subject.

By way of example only, standard chemotherapy treatments for metastaticcolorectal cancer are described herein below.

In one preferred embodiment, the methods of the invention are used totreat colorectal cancer including metastatic colorectal cancer.Colorectal cancer is the third most common cause of cancer mortality inthe United States. It was estimated that approximately 129,000 new casesof colorectal cancer would be diagnosed and 56,000 deaths would occurdue to colorectal cancer in the United States in 1999, Landis et al.,Cancer J Clin. 49:8-31 (1999). Approximately 70% of colorectal cancerpatients present with disease that is potentially curable by surgicalresection, August et al., Cancer Metastasis Rev 3:303-24 (1984).However, the prognosis for the 30% who present with advanced ormetastatic disease and for the 20% who relapse following resection ispoor. The median survival for those with metastatic disease is 12-14months, Advanced Colorectal Cancer Meta-Analysis Project, J Clin Oncol10:896-903 (1992).

The standard treatment for metastatic colorectal cancer in the UnitedStates has been until recently chemotherapy with 5-fluorouracil (5-FU)plus a biochemical modulator of 5-FU, leucovorin, Advanced ColorectalCancer Meta-Analysis Project, J Clin Oncol 10:896-903 (1992); Moertel NEngl J Med 330:1136-42 (1994). The combination of 5-FU/leucovorinprovides infrequent, transient shrinkage of colorectal tumors but hasnot been demonstrated to prolong survival compared with 5-FU alone(Advanced Colorectal Cancer Meta-Analysis Project, J Clin Oncol10:896-903 (1992)), and 5-FU has not been demonstrated to prolongsurvival compared with an ineffective therapy plus best supportive care,Ansfield et al. Cancer 39:34-40 (1977). The lack of a demonstratedsurvival benefit for 5-FU/leucovorin may be due in part to inadequatelysized clinical trials. In a large randomized trial of patients receivingadjuvant chemotherapy for resectable colorectal cancer, 5-FU/leucovorindemonstrated prolonged survival compared with lomustine (MeCCNU),vincristine, and 5-FU (MOF; Wolmark et al. J Clin Oncol 11:1879-87(1993).

In the United States, 5-FU/leucovorin chemotherapy is commonlyadministered according to one of two schedules: the Mayo Clinic andRoswell Park regimens. The Mayo Clinic regimen consists of an intensivecourse of 5-FU plus low-dose leucovorin (425 mg/m 2 5-FU plus 20 mg/m 2leucovorin administered daily by intravenous [IV] push for 5 days, withcourses repeated at 4- to 5-week intervals), Buroker et al. J Clin Oncol12:14-20 (1994). The Roswell Park regimen consists of weekly 5-FU plushigh-dose leucovorin (500-600 mg/m 2 5-FU administered by IV push plus500 mg/m 2 leucovorin administered as a 2-hour infusion weekly for 6weeks, with courses repeated every 8 weeks), Petrelli et al., J ClinOncol 7:1419-26 (1989). Clinical trials comparing the Mayo Clinic andRoswell Park regimens have not demonstrated a difference in efficacy buthave been underpowered to do so, Buroker et al., J Clin Oncol 12:14-20(1994); Poon et al., J Clin Oncol 7:1407-18 (1989). The toxicityprofiles of the two regimens are different, with the Mayo Clinic regimenresulting in more leukopenia and stomatitis and the Roswell Park regimenresulting in more frequent diarrhea. Patients with newly diagnosedmetastatic colorectal cancer receiving either regimen can expect amedian time to disease progression of 4-5 months and a median survivalof 12-14 months, Petrelli et al., J Clin Oncol 7:1419-26 (1989);Advanced Colorectal Cancer Meta—Analysis Project, J Clin Oncol10:896-903 (1992); Buroker et al., J Clin Oncol 12:14-20 (1994); Cocconiet al., J Clin Oncol 16:2943-52 (1998).

Recently, a new first-line therapy for metastatic colorectal cancer hasemerged. Two randomized clinical trials, each with approximately 400patients, evaluated irinotecan in combination with 5-FU/leucovorin,Saltz et al., Proc ASCO 18:233a (1999); Douillard et al., Lancet355:1041-7 (2000). In both studies, the combination ofirinotecan/5-FU/leucovorin demonstrated statistically significantincreases in survival (of 2.2 and 3.3 months), time to diseaseprogression and response rates as compared with 5-FU/leucovorin alone.The benefits of irinotecan came at a price of increased toxicity:addition of irinotecan to 5-FU/leucovorin was associated with anincreased incidence of National Cancer Institute Common ToxicityCriteria (NCI-CTC) Grade 3/4 diarrhea, Grade 3/4 vomiting, Grade 4neutropenia, and asthenia compared with 5-FU/leucovorin alone. There isalso evidence showing that single-agent irinotecan prolongs survival inthe second-line setting, Cunningham et al., Lancet 352:1413-18 (1998);Rougier et al., Lancet 352:1407-12 (1998). Two randomized studies havedemonstrated that irinotecan prolongs survival in patients who haveprogressed following 5-FU therapy. One study compared irinotecan to bestsupportive care and showed a 2.8-month prolongation of survival; theother study compared irinotecan with infusional 5-FU and showed a2.2-month prolongation of survival. The question of whether irinotecanhas more effect on survival in the first- or second-line setting has notbeen studied in a well-controlled fashion.

Dosage and Administration

The antibodies and chemotherapeutic agents of the invention areadministered to a human patient, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

In one embodiment, the treatment of the present invention involves thecombined administration of an anti-VEGF antibody and one or morechemotherapeutic agents. The present invention contemplatesadministration of cocktails of different chemotherapeutic agents. Thecombined administration includes coadministration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992). The chemotherapeutic agent may precede, or followadministration of the antibody or may be given simultaneously therewith.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.In a combination therapy regimen, the compositions of the presentinvention are administered in a therapeutically effective or synergisticamount. As used herein, a therapeutically effective amount is such thatco-administration of anti-VEGF antibody and one or more othertherapeutic agents, or administration of a composition of the presentinvention, results in reduction or inhibition of the targeting diseaseor condition. A therapeutically synergistic amount is that amount ofanti-VEGF antibody and one or more other therapeutic agents necessary tosynergistically or significantly reduce or eliminate conditions orsymptoms associated with a particular disease.

Depending on the type and severity of the disease, about 1 μg/kg to 50mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. In a preferred aspect, theantibody of the invention is administered every two to three weeks, at adose ranged from about 5 mg/kg to about 15 mg/kg. More preferably, suchdosing regimen is used in combination with a chemotherapy regimen as thefirst line therapy for treating metastatic colorectal cancer. In someaspects, the chemotherapy regimen involves the traditional high-doseintermittent administration. In some other aspects, the chemotherapeuticagents are administered using smaller and more frequent doses withoutscheduled breaks (“metronomic chemotherapy”). The progress of thetherapy of the invention is easily monitored by conventional techniquesand assays.

Further information about suitable dosages is provided in the Examplebelow.

Efficacy of the Treatment

The main advantage of the treatment of the present invention is theability of producing marked anti-cancer effects in a human patientwithout causing significant toxicities or adverse effects, so that thepatient benefited from the treatment overall. The efficacy of thetreatment of the invention can be measured by various endpoints commonlyused in evaluating cancer treatments, including but not limited to,tumor regression, tumor weight or size shrinkage, time to progression,duration of survival, progression free survival, overall response rate,duration of response, and quality of life. Because the anti-angiogenicagents of the invention target the tumor vasculature and not necessarilythe neoplastic cells themselves, they represent a unique class ofanticancer drugs, and therefore may require unique measures anddefinitions of clinical responses to drugs. For example, tumor shrinkageof greater than 50% in a 2-dimensional analysis is the standard cut-offfor declaring a response. However, the anti-VEGF antibody of theinvention may cause inhibition of metastatic spread without shrinkage ofthe primary tumor, or may simply exert a tumouristatic effect.Accordingly, novel approaches to determining efficacy of ananti-angiogenic therapy should be employed, including for example,measurement of plasma or urinary markers of angiogenesis and measurementof response through radiological imaging.

In one embodiment, the present invention can be used for increasing theduration of survival of a human patient susceptible to or diagnosed witha cancer. Duration of survival is defined as the time from firstadministration of the drug to death. In a preferred aspect, theanti-VEGF antibody of the invention is administered to the human patientin combination with one or more chemotherapeutic agents, thereby theduration of survival of the patient is effectively increased as comparedto a chemotherapy alone. For example, patient group treated with theanti-VEGF antibody combined with a chemotherapeutic cocktail of at leasttwo, preferably three, chemotherapeutic agents may have a medianduration of survival that is at least about 2 months, preferably betweenabout 2 and about 5 months, longer than that of the patient grouptreated with the same chemotherapeutic cocktail alone, said increasebeing statistically significant. Duration of survival can also bemeasured by stratified hazard ratio (HR) of the treatment group versuscontrol group, which represents the risk of death for a patient duringthe treatment. Preferably, a combination treatment of anti-VEGF antibodyand one or more chemotherapeutic agents significantly reduces the riskof death by at least about 30% (i.e., a stratified HR of about 0.70),preferably by at least about 35% (i.e., a stratified HR of about 0.65),when compared to a chemotherapy alone.

In another embodiment, the present invention provides methods forincreasing progression free survival of a human patient susceptible toor diagnosed with a cancer. Time to disease progression is defined asthe time from administration of the drug until disease progression. In apreferred embodiment, the combination treatment of the invention usinganti-VEGF antibody and one or more chemotherapeutic agents significantlyincreases progression free survival by at least about 2 months,preferably by about 2 to about 5 months, when compared to a treatmentwith chemotherapy alone.

In yet another embodiment, the treatment of the present inventionsignificantly increases response rate in a group of human patientssusceptible to or diagnosed with a cancer who are treated with varioustherapeutics. Response rate is defined as the percentage of treatedpatients who responded to the treatment. In one aspect, the combinationtreatment of the invention using anti-VEGF antibody and one or morechemotherapeutic agents significantly increases response rate in thetreated patient group compared to the group treated with chemotherapyalone, said increase having a Chi-square p-value of less than 0.005.

In one aspect, the present invention provides methods for increasingduration of response in a human patient or a group of human patientssusceptible to or diagnosed with a cancer. Duration of response isdefined as the time from the initial response to disease progression. Ina combination treatment of the invention using anti-VEGF antibody andone or more chemotherapeutic agents, a statistically significantincrease of at least 2 months in duration of response is obtainable andpreferred.

Safety of the Treatment

The present invention provides methods of effectively treating cancerswithout significant adverse effects to the human patient subject totreatment. The clinical outcomes of the treatment according to theinvention are somewhat unexpected, in that several adverse eventsthought to be associated with anti-angiogenic therapies are not observedduring the course of treatments according to the present invention. Forexample, previous clinical studies suggested that treatment withanti-VEGF antibodies may cause thrombosis (fatal in certain case),hypertension, proteinuria and epistaxis (bleeding). However, combinationtherapy of the invention using anti-VEGF antibody combined with achemotherapy cocktail comprising at least two, preferably three,chemotherapeutic agents does not significantly increase incidentoccurrences of these adverse events, when compared with the chemotherapyalone. Thus, the treatment of the present invention unexpectedlycontains side effects at acceptable level, at the same timesignificantly improve anticancer efficacy.

V. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container, alabel and a package insert. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is ananti-VEGF antibody. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes. In addition, the article of manufacture comprises a packageinserts with instructions for use, including for example a warning thatthe composition is not to be used in combination with anthacycline-typechemotherapeutic agent, e.g. doxorubicin, or epirubicin, or instructingthe user of the composition to administer the anti-VEGF antibodycomposition and an antineoplastic composition to a patient.

Deposit of Materials

The following hybridoma cell line has been deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC), Manassas, Va., USA:

Antibody Designation ATCC No. Deposit Date A4.6.1 ATCC HB-10709 Mar. 29,1991

The following examples are intended merely to illustrate the practice ofthe present invention and are not provided by way of limitation. Thedisclosures of all patent and scientific literatures cited herein areexpressly incorporated in their entirety by reference.

VI. Examples Example 1 Addition of an Anti-VEGF Antibody to BolusIrinotecan/Fluorouracil/Leucovorin (IFL) in First Line MetastaticColorectal Cancer

A multicenter, Phase III, randomized, active-controlled trial wasconducted to evaluate the efficacy and safety of bevacizumab when addedto standard first-line chemotherapy used to treat metastatic colorectalcancer. The trial enrolled over 900 patients with histologicallyconfirmed, previously untreated, bi-dimensionally measurable metastaticcolorectal cancer.

Methods and Materials Anti-VEGF Antibody Bevacizumab

The anti-VEGF antibody “Bevacizumab (BV)”, also known as “rhuMAb VEGF”or “Avastin™”, is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. U.S. Pat. No. 6,582,959; WO 98/45331. Approximately 93% ofthe amino acid sequence of bevacizumab, including most of the frameworkregions, is derived from human IgG1, and about 7% of the sequence isderived from the murine antibody A4.6.1. Bevacizumab has a molecularmass of about 149,000 daltons and is glycosylated.

Identities of the polypeptide and sites of glycosylation were deducedfrom the amino acid composition and peptide map. The size and chargecharacteristics of the molecule and the purity of the clinical lots weredemonstrated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis or capillary electrophoresis non-gel sieving,isoelectric focusing, as well as ion-exchange and size-exclusionchromatography. The activity of bevacizumab was quantified by a bindingenzyme-linked immunosorbent assay or a kinase receptor assay forrecombinant human VEGF.

bevacizumab was produced by recombinant DNA technology, using agenetically engineered Chinese hamster ovary cell line. The protein waspurified from the cell culture medium by routine methods of columnchromatography and filtration. The final product was tested for quality,identity, safety, purity, potency, strength, and excipient/chemicalcomposition according to U.S. Food and Drug Administration guidelines.The purity of bevacizumab is >95%. bevacizumab is supplied as a clear toslightly opalescent, sterile liquid ready for parenteral administration.

Patient Selection

Eligible patients had histologically confirmed metastatic colorectalcarcinoma, with bidimensionally measurable disease. Other inclusioncriteria included an age of at least 18 years, an Eastern CooperativeOncology Group (ECOG) performance status of 0 or 1 (Oken et al. (1982)Am. J. Clin. Oncol. 5:649-55), a life expectancy of more than threemonths, and written informed consent. Adequate hematologic, hepatic, andrenal function (including urinary excretion of no more than 500 mg ofprotein per day) was also required.

Exclusion criteria included prior chemotherapy or biologic therapy formetastatic disease (adjuvant or radiosensitizing use offluoropyrimidines with or without leucovorin or levamisole more than 12months before study entry was permitted), receipt of radiotherapy within14 days before the initiation of study treatment, major surgery within28 days before the initiation of study treatment, clinically significantcardiovascular disease, clinically detectable ascites, pregnancy orlactation, regular use of aspirin (more than 325 mg per day) or othernonsteroidal and inflammatory agents, preexisting bleeding diatheses orcoagulopathy or the need for full-dose anticoagulation, and knowncentral nervous system metastases.

Study Design

Eligible patients were assigned to treatment with the use of a dynamicrandomization algorithm that was designed to achieve overall balancebetween groups; randomization was stratified according to study center,baseline ECOG performance status (0 vs. 1), site of primary disease(colon vs. rectum), and number of metastatic sites (one vs. more thanone). Initially, patients were randomly assigned in a 1:1:1 ratio toreceive IFL plus placebo, IFL plus bevacizumab, or fluorouracil andleucovorin plus bevacizumab (Table 1), each of which was to continueuntil disease progression or unacceptable adverse effects occurred orfor a maximum of 96 weeks.

TABLE 1 First-Line Treatment Regimens* Treatment Starting Dose ScheduleIrinotecan 125 mg/m² of Once weekly for 4 wk; cycle body-surface arearepeated every 6 wk Fluorouracil 500 mg/m² Leucovorin 20 mg/m² PlaceboEvery 2 wk Irinotecan 125 mg/m² Once weekly for 4 wk; Fluorouracil 500mg/m² cycle repeated every 6 wk Leucovorin 20 mg/m² Bevacizumab 5 mg/kgof Every 2 wk body weight Fluorouracil 500 mg/m² Once weekly for 4 wk;Leucovorin 500 mg/m² cycle repeated every 8 wk Bevacizumab 5 mg/kg Every2 wk *Treatment with fluorouracil, leucovorin, and bevacizumab wasdiscontinued after the safety of adding bevacizumab to the regimen ofirinotecan, fluorouracil, and leucovorin was confirmed. Confirmationoccurred after the randomization of 313 patients. All drugs were givenintravenously.

An interim analysis was scheduled to be performed after 300 patientsunderwent randomization, at which time an unblinded, independentdata-monitoring committee was to assess the safety of IFL plusbevacizumab, on the basis of all the available safety information,including the number of deaths in each group, but in the absence ofinformation related to tumor response. If the data-monitoring committeefound no untoward adverse events attributable to the addition ofbevacizumab to IFL, the enrollment of patients in the group assigned toreceive fluorouracil and leucovorin plus bevacizumab was to bediscontinued, and additional patients would be randomly assigned in a1:1 ratio to receive either IFL plus placebo or IFL plus bevacizumab.However, if the data-monitoring committee concluded that the safetyprofile of TFL plus bevacizumab was unacceptable, assignment to thattreatment was to be discontinued, and patients would instead be randomlyassigned in a 1:1 ratio to receive either the combination offluorouracil and leucovorin plus bevacizumab or IFL plus placebo.

Tumor responses and progression were determined with the use of theResponse Evaluation Criteria in Solid Tumors. Therasse et al. (2000) J.Natl. Cancer Inst. 92:205-16. At the time of disease progression, thetreatment assignment was revealed and patients could be offeredsecond-line treatment. Such patients in the group assigned tobevacizumab-containing treatment had the option to continue bevacizumabduring this second-line treatment. No crossovers were allowed in thegroup given IFL plus placebo. Patients assigned to a treatmentcontaining bevacizumab who had no signs of progressive disease at theend of the 96-week study period could continue to receive bevacizumab ina separate extension study. Patients in a group receiving bevacizumabwho had a confirmed complete response or unacceptable adverse effectsfrom chemotherapy could discontinue chemotherapy and receive bevacizumabalone.

Bevacizumab (or placebo) was administered concomitantly withchemotherapy. Doses of bevacizumab and chemotherapy were recalculated ifa patient's weight changed by at least 10 percent during the study.Standard intracycle and intercycle dose modifications of irinotecan andfluorouracil (according to the package insert)^(‘∘) were permitted inpatients with treatment-related adverse events. The doses of leucovorinand bevacizumab were not altered.

In the analysis of survival and subsequent treatment, all patients werefollowed until death, loss to follow-up, or termination of the study.

Assessments

After the baseline evaluation, tumor status was assessed every 6 weeksfor the first 24 weeks of the study and then every 12 weeks for theremainder of therapy. All complete and partial responses requiredconfirmation at least four weeks after they were first noted.

Safety was assessed on the basis of reports of adverse events,laboratory-test results, and vital sign measurements. Adverse eventswere categorized according to the Common Toxicity Criteria of theNational Cancer Institute, version 2, in which a grade of 1 indicatesmild adverse events, a grade of 2 moderate adverse events, a grade of 3serious adverse events, and a grade of 4 life-threatening adverseevents. Prespecified safety measures included the incidence of alladverse events, all serious adverse events, and adverse events that havebeen associated with bevacizumab—hypertension, thrombosis, bleeding ofgrade 3 or 4, and proteinuria—as well as diarrhea of grade 3 or 4, andchanges from baseline in various laboratory values and vital signs.

To monitor the safety of the regimen of IFL plus placebo and of IFL plusbevacizumab, the incidence of death, serious adverse events, diarrhea ofgrade 3 or 4, bleeding of grade 3 or 4 from any source, and thrombosiswas monitored during the study in an un-blinded fashion by thedata-safety monitoring committee until the completion of recruitment orthe time of the interim analysis of efficacy, whichever came first.

Statistical Analysis

The primary outcome measure was the duration of overall survival;survival was measured without regard to subsequent treatments. There wasno crossover between groups, however. Survival analysis techniques suchas the Kaplan-Meier method, log-rank test, and Cox proportional hazardsmodel were used. Secondary outcome measures were progression-freesurvival, objective response rates (complete and partial responses), theduration of responses, and the quality of life.

For patients who were alive at the time of analysis, data on survivalwere censored at the time of the last contact. Progression-free survivalwas defined as the time from randomization to progression or deathduring the study, with death during the study defined as any death thatoccurred within 30 days after the last dose of bevacizumab orchemotherapy. For patients without disease progression at the time ofthe final analysis, data on progression-free survival were censored atthe last assessment of tumor status or on day 0 if no further assessmentwas performed after baseline. Patients without adequate follow-up datawere categorized as having no response.

To detect a hazard ratio of 0.75 for death in the group given IFL plusbevacizumab as compared with the control group, approximately 385 deathswere required. All calculations were performed with the log-rank testand involved two-sided P values, with an alpha value of 0.05, astatistical power of 80 percent, and one interim analysis of efficacy.

Interim analyses were conducted in an un-blinded fashion. An interimanalysis of safety was conducted after the random assignment ofapproximately 100 patients to each group. A second interim analysis ofsafety and efficacy was performed after 193 deaths had occurred (halfthe number of required events).

Efficacy analyses were performed according to the intention-to-treatprinciple. Safety analyses included all patients who received at leastone dose of study medication.

Results Characteristics of the Patients

During a period of about twenty months, 923 patients underwentrandomization at 164 sites in the United States, Australia, and NewZealand. After 313 patients had been randomly assigned to one of thethree groups—100 to IFL plus placebo, 103 to IFL plus bevacizumab, and110 to fluorouracil, leucovorin, and bevacizumab—assignment to the groupgiven fluorouracil, leucovorin, and bevacizumab was halted (the resultsin this group are not reported). This step was required by the protocolafter the first formal interim analysis of safety concluded that theregimen of IFL plus bevacizumab had an acceptable safety profile andthat assignment to this group could continue.

The intention-to-treat analysis of the primary end point of overallsurvival included 411 patients in the group given IFL plus placebo and402 patients in the group given IFL plus bevacizumab. Table 2 showsselected demographic and baseline characteristics, which were wellbalanced between the groups. Similar numbers of patients in each grouphad previously undergone surgery or received radiation therapy oradjuvant chemotherapy for colorectal cancer.

Treatment

The median duration of therapy was 27.6 weeks in the group given IFLplus placebo and 40.4 weeks in the group given IFL plus bevacizumab. Thepercentage of the planned dose of irinotecan that was given was similarin the two groups (78 percent in the group given IFL plus placebo and 73percent in the group given IFL plus bevacizumab).

As of the date of data cutoff, 33 patients in the group given IFL plusplacebo and 71 in the group given TFL plus bevacizumab were still takingtheir assigned initial therapy. The rates of use of second-linetherapies that may have affected survival, such as oxaliplatin ormetastasectomy, were well balanced between the two groups. In bothgroups, approximately 50 percent of patients received some form ofsecond line therapy; 25 percent of all patients received oxaliplatin,and less than 2 percent of patients underwent metastasectomy.

TABLE 2 Selected Demographic and Baseline Characteristics.* IFL plusPlacebo IFL plus Bevacizumab Characteristic (N = 411) (N = 402) Sex (%)MALE 60 59 FEMALE 40 41 MEAN AGE (YR) 59.2 59.5 Race (%) White 80 79Black 11 12 Other 9 9 Location of center (%) United States 99 99Australia or New Zealand <1 <1 ECOG performance status (%)  0 55 58  144 41  2 <1 <1 Type of cancer (%) Colon 81 77 Rectal 19 23 Number ofmetastatic sites (%)  1 39 37 >1 61 63 Prior cancer therapy (%) Adjuvantchemotherapy 28 24 Radiation therapy 14 15 Median duration of 4 4metastatic disease (mo) *There were no significant differences betweengroups. IFL denotes irinotecan, fluorouracil, and leucovorin, and ECOGEastern Cooperative Oncology Group.

Efficacy

The median duration of overall survival, the primary end point, wassignificantly longer in the group given IFL plus bevacizumab than in thegroup given IFL plus placebo (20.3 months vs. 15.6 months), whichcorresponds to a hazard ratio for death of 0.66 (P<0.001) (Table 3 andFIG. 1), or a reduction of 34 percent in the risk of death in thebevacizumab group. The one-year survival rate was 74.3 percent in thegroup given IFL plus bevacizumab and 63.4 percent in the group given IFLplus placebo (P<0.001). In the subgroup of patients who receivedsecond-line treatment with oxaliplatin, the median duration of overallsurvival was 25.1 months in the group given IFL plus bevacizumab and22.2 months in the group given IFL plus placebo.

The addition of bevacizumab to IFL was associated with increases in themedian duration of progression-free survival (10.6 months vs. 6.2months; hazard ratio for progression, 0.54, for the comparison with thegroup given IFL plus placebo; P<0.001); response rate (44.8 percent vs.34.8 percent; P=0.004); and the median duration of response (10.4 monthsvs. 7.1 months; hazard ratio for progression, 0.62; P=0.001) (Table 3).FIG. 2 shows the Kaplan-Meier estimates of progression free survival.Treatment effects were consistent across prespecified subgroups,including those defined according to age, sex, race, ECOG performancestatus, location of the primary tumor, presence or absence of prioradjuvant therapy, duration of metastatic disease, number of metastaticsites, years since the diagnosis of colorectal cancer, presence orabsence of prior radiotherapy, baseline tumor burden, and serumconcentrations of albumin, alkaline phosphatase, and lactatedehydrogenase.

TABLE 3 Analysis of Efficacy* IFL plus IFL plus End Point PlaceboBevacizumab P Value Median survival (mo) 15.6 20.3 <0.001 Hazard ratiofor death 0.66 One-year survival rate (%) 63.4 74.3 <0.001Progression-free survival (mo) 6.2 10.6 <0.001 Hazard ratio forprogression 0.54 Overall response rate (%) 34.8 44.8 0.004 Completeresponse 2.2 3.7 Partial response 32.6 41.0 Median duration of response(mo) 7.1 10.4 0.001 Hazard ratio for relapse 0.62 *IFL denotesirinotecan, fluorouracil, and leucovorin.

Safety

Table 4 presents the incidence of selected grade 3 or 4 adverse eventsduring the assigned treatment, without adjustment for the medianduration of therapy (27.6 weeks in the group given IFL plus placebo and40.4 weeks in the group given IFL plus bevacizumab). The incidence ofany grade 3 or 4 adverse events was approximately 10 percentage pointshigher among patients receiving IFL plus bevacizumab than among patientsreceiving IFL plus placebo, largely because of an increase in theincidence of grade 3 hypertension (requiring treatment) and smallincreases in the incidence of grade 4 diarrhea and leukopenia. However,there was no significant difference in the incidence of adverse eventsleading to hospitalization or to the discontinuation of study treatmentor in the 60-day rate of death from any cause.

TABLE 4 Selected Adverse Events.* IFL plus IFL plus Placebo Bevacizumab(N = 397) (N = 393) Adverse Event percent Any grade 3 or 4 adverse event74.0 84.9** Adverse event leading to hospitalization 39.6 44.9 Adverseevent leading to discontinuation of 7.1 8.4 treatment Adverse eventleading to death 2.8 2.6 Death within 60 days 4.9 3.0 Grade 3 or 4leukopenia 31.1 37.0 Hypertension Any 8.3 22.4** Grade 3 2.3 11.0** Anythrombotic event 16.2 19.4 Deep thrombophletitis 6.3 8.9 Pulmonaryembolus 5.1 3.6 Grade 3 or 4 bleeding 2.5 3.1 Proteinuria Any 21.7 26.5Grade 2 5.8 3.1 Grade 3 0.8 0.8 Gastrointestinal perforation 0.0 1.5*Data were not adjusted for differences in the median duration oftherapy between the group given irinotecan, fluorouracil, and leucovorin(IFL) plus placebo and the group given IFL plus bevacizumab (27.6 weeksvs. 40.4 weeks). **P < 0.01. Only patients who received at least onestudy-drug treatment are included.

Phase 1 and 2 trials had identified hemorrhage, thromboembolism,proteinuria, and hypertension as possible bevacizumab-associated adverseeffects. However, in the present study, only the incidence ofhypertension was clearly increased in the group given IFL plusbevacizumab, as compared with the group given IFL plus placebo. Allepisodes of hypertension were manageable with standard oralantihypertensive agents (e.g., calcium-channel blockers,angiotensin-converting-enzyme inhibitors, and diuretics). There were nodiscontinuations of bevacizumab therapy, hypertensive crises, or deathsrelated to hypertension in the bevacizumab group.

Rates of grade 2 or 3 proteinuria (there were no episodes of grade 4proteinuria or nephrotic syndrome) and grade 3 or 4 bleeding from anycause were similar in the two groups, although all three cases of grade4 bleeding were in the group given IFL plus bevacizumab. The incidenceof all venous and arterial thrombotic events was 19.4 percent in thegroup given IFL plus bevacizumab and 16.2 percent in the group given IFLplus placebo (P=0.26).

Gastrointestinal perforation occurred in six patients (1.5 percent)receiving IFL plus bevacizumab. One patient died as a direct result ofthis event, whereas the other five recovered (three of them were able torestart treatment without subsequent complications). Of the six patientswith a perforation, three had a confirmed complete or partial responseto IFL plus bevacizumab. Factors other than the study treatment that mayhave been associated with gastrointestinal perforation were colonsurgery within the previous two months in two patients and peptic-ulcerdisease in one patient.

The results of this phase III study provide direct support for a broadlyapplicable use of antiangiogenic agents in the treatment of cancer. Theaddition of bevacizumab, an anti-VEGF antibody, to IFL chemotherapyconferred a clinically meaningful and statistically significantimprovement in cancer patients as measured by, for example, overallsurvival, progression-free survival, response rate and duration ofresponse. The increase of 4.7 months in the median duration of survivalattributable to bevacizumab is as large as or larger than that observedin any other phase 3 trial for the treatment of colorectal cancer.Goldberg et al. (2004) J. Clin. Oncol. 22:23-30. The median survival of20.3 months in the bevacizumab-treated population occurred in spite ofthe limited availability of oxaliplatin for second-line therapy duringthis trial.

As compared with IFL alone, the regimen of IFL plus bevacizumabincreased progression-free survival from a median of 6.2 months to 10.6months, the overall response rate from 34.8 percent to 44.8 percent, andthe median duration of response from 7.1 months to 10.4 months. Theseimprovements are clinically meaningful. It was not predicted that theabsolute improvement in the response rate of 10 percent with IFL plusbevacizumab would have been associated with an increase in survival ofthis magnitude. This observation suggests that the primary mechanism ofbevacizumab is the inhibition of tumor growth, rather thancytoreduction.

This clinical benefit was accompanied by a relatively modest increase inside effects of treatment, which were easily managed. There was anabsolute increase of approximately 10 percent in the overall incidenceof grade 3 and 4 adverse effects, attributable largely to hypertensionrequiring treatment, diarrhea, and leukopenia. The 60-day rates of deathfrom any cause, hospitalization, and discontinuation of treatment werenot significantly increased by the addition of bevacizumab to IFL.

Previous phase 1 and 2 clinical trials suggested that treatment withbevacizumab alone or with chemotherapy resulted in an increasedincidence of thrombosis, bleeding, proteinuria, and hypertension.Kabbinavar et al. (2003) J. Clin. Oncol. 21:60-65; Yang et al. (2003)New Engl. J. Med. 349:427-34. With the exception of hypertension, anexcess of these side effects was not found as compared with theirincidence in the group given IFL plus placebo—thus highlighting theimportance of randomized, placebo-controlled studies for the evaluationof safety as well as efficacy. One new potential adverse effect thatoccurred was gastrointestinal perforation. This complication wasuncommon and had variable clinical presentations. Severe bowelcomplications, particularly in patients with neutropenia, have beenreported with IFL and other chemotherapy regimens for colorectal cancerand in one series, fistulas were reported in over 2 percent of patientstreated with fluorouracil-based regimens. Saltz et al. (2000) New Engl.J. Med. 343:905-914; Rothenberg et al. (2001) J. Clin. Oncol. 19:3801-7;Tebbutt et al. (2003) Gut 52:568-73. No such events occurred in thegroup given IFL plus placebo, whereas six cases were observed in thegroup given IFL plus bevacizumab (1.5 percent), sometimes in the settingof overall tumor responses. Although three of these six patients wereable to restart treatment without subsequent complications, one patientdied and two discontinued therapy permanently as a result of thiscomplication.

While previous animal studies and early phase clinical trials havesuggested uses of anti-angiogenic therapy for treating cancer, thepresent study showed for the first time that using an angiogenicinhibitor, such as an anti-VEGF antibody, indeed results instatistically significant and clinically meaningful benefits for cancerpatients.

Example 2 Addition of Bevacizumab to Bolus 5-FU/Leucovorin in First-LineMetastatic Colorectal Cancer

This randomized, phase II trial compared bevacizumab plus 5-fluorouraciland leucovorin (5-FU/LV) versus placebo plus 5-FU/LV as first-linetherapy in patients considered non-optimal candidates for first-lineirinotecan.

Patients and Methods Patient Eligibility

Patients with histologically confirmed, previously untreated, measurablemetastatic colorectal cancer were eligible if, in the judgment of theinvestigator, they were not optimal candidates for first-lineirinotecan-containing therapy and had at least one of the followingcharacteristics: age above 65 years, ECOG PS of 1 or 2, serum albuminequal or less than 3.5 g/dL, or prior radiotherapy to abdomen or pelvis.Patients were excluded if they had undergone major surgical proceduresor open biopsy, or had experienced significant traumatic injury, within28 days prior to study entry; anticipated need for major surgery duringthe course of the study; were currently using or had recently usedtherapeutic anticoagulants (except as required for catheter patency),thrombolytic therapy or chronic, daily treatment with aspirin 325mg/day) or nonsteroidal anti-inflammatory medications; had a serious,non-healing wound, ulcer, or bone fracture; had a history or evidence ofCNS metastases; were pregnant or lactating; or had proteinuria orclinically significant impairment of renal function at baseline. Allpatients provided written informed consent for their participation.

Study Design and Treatments

An interactive voice response system was used to randomly assigneligible patients to one of two treatment groups: 5-FU/LV plus placeboor 5-FU/LV plus bevacizumab. A dynamic randomization algorithm wasutilized to achieve balance overall and within each of the followingcategories: study center, baseline ECOG performance status (0 vs.≧1),site of primary disease (colon vs. rectum), and number of metastaticsites (1 vs.>1). The 5-FU/LV treatment, comprising LV 500 mg/m² over 2hours and 5-FU 500 mg/m² as a bolus midway through the LV infusion(Roswell Park regimen; Petrelli et al. (1989) J. Clin. Oncol.7:1419-1426), was administered weekly for the first 6 weeks of each8-week cycle. Chemotherapy was continued until study completion (96weeks) or disease progression. Bevacizumab 5 mg/kg or placebo wasadministered every 2 weeks. Patients in the bevacizumab arm who had aconfirmed complete response or experienced unacceptable toxicity as aresult of chemotherapy treatment were allowed to discontinue 5-FU/LV andcontinue receiving bevacizumab alone as first-line treatment. At thetime of disease progression, patients were unblinded to their treatmentassignment and could receive any second-line treatment at the discretionof the investigator. Only patients who had been randomized to thebevacizumab group could receive bevacizumab as a component ofsecond-line treatment. After completing the study, patients werefollowed for any subsequent treatment and survival every 4 months untildeath, loss to follow-up, or termination of the study.

Study Assessments

Patients underwent an assessment of tumor status at baseline and atcompletion of every 8-week cycle using appropriate radiographictechniques, typically spiral CT scanning. Tumor response, orprogression, was determined by both the investigator and an independentradiology facility (IRF) utilizing the Response Evaluation Criteria inSolid Tumors. Therasse et al. (2000). The IRF assessment was performedwithout knowledge of the treatment assignment or investigatorassessment. In addition, patients completed the Functional Assessment ofCancer Therapy—Colorectal (FACT-C), Version 4, a validated instrumentfor assessing quality of life (QOL) in colorectal cancer patients, atbaseline and prior to each treatment cycle until disease progression.Ward et al. (1999) Qual. Life Res. 8:181-195.

Safety was assessed from reports of adverse events, laboratory testresults, and vital sign measurements. Adverse events and abnormallaboratory results were categorized using the National Cancer InstituteCommon Toxicity Criteria (NCI-CTC), Version 2. Prespecified safetymeasures included four adverse events of special interest (hypertension,proteinuria, thrombosis, and bleeding) based on findings of previousclinical trials of bevacizumab.

Statistical Analysis

The primary outcome measure was duration of overall survival. Secondaryoutcome measures included progression-free survival, objective responserate (complete and partial), response duration, and change in the FACT-CQOL score. Survival duration was defined as the time from randomizationto death. For patients alive at the time of analysis, duration ofsurvival was censored at the date of last contact. Progression-freesurvival was defined as the time from randomization to the earlier ofdisease progression or death on study, defined as death from any causewithin 30 days of the last dose of study drug or chemotherapy. Forpatients alive without disease progression at the time of analysis,progression-free survival was censored at their last tumor assessment,or day 1 (the first day of study treatment) if no postbaselineassessment was performed. In the analysis of objective response,patients without tumor assessments were categorized as nonresponders.Disease progression and response analyses were based on the IRFassessments. Change in quality of life was analyzed as time todeterioration in QOL (TDQ), defined as the length of time fromrandomization to a the earliest of a ≧3-point decrease from baseline incolon-cancer specific FACT-C subscale score (CCS), disease progression,or death on study. TDQ was also determined for the TOI-C (sum of CCS,physical and functional well-being) and total FACT-C for changes frombaseline of 7 and 9 points, respectively.

To detect a hazard ratio of 0.61 for death in the 5-FU/LV/bevacizumabgroup relative to the 5-FU/LV/placebo group, approximately 133 deathswere required. A two-tailed, log-rank test at the 0.05 level ofsignificance with 80% power and two interim analyses were assumed in thecalculations. Interim analyses were conducted by an unblinded,independent Data Monitoring Committee (DMC). A safety interim analysiswas conducted after 44 deaths and a second safety and efficacy interimanalysis was conducted after 89 deaths. The interim efficacy analysiswas governed by a formal group sequential stopping rule based on anO'Brien-Fleming spending function. Kaplan-Meier methodology was appliedto estimate the median survival, progression free survival, and durationof response time for each treatment group. Hazard ratios for thebevacizumab group relative to the placebo group were determined usingthe stratified Cox proportional hazards model. A two-sided stratifiedlog rank test was used to compare the two groups. Stratified analysesincluded baseline ECOG performance status, site of primary disease, andthe number of metastatic sites. Objective response rates were comparedby the Chi-squared test. As exploratory analyses, the Cox proportionalhazards model was used to estimate the effect of risk factors onmodifications of treatment effect for duration of survival andprogression-free survival. Efficacy analyses were performed on theintent-to-treat population, defined as all randomized patients. Safetyanalyses included all patients who received at least one dose of studydrug.

Results Patient Characteristics

In a period of twenty three months, 209 patients were randomized at 60sites in the United States and Australia/New Zealand. For theintent-to-treat analysis of the primary endpoint (overall survival),there were 105 patients in the 5-FU/LV/placebo group and 104 in the5-FU/LV/bevacizumab group. Selected demographic and baselinecharacteristics similar to those described in Example 1 were reasonablybalanced between treatment groups. Low serum albumin (≦3.5 g/dL) atbaseline was less common in the bevacizumab group than in the placebogroup.

Treatment

The median duration of therapy was 23 weeks in the 5-FU/LV/placebo groupand 31 weeks in the 5-FU/LV/bevacizumab group, and the 5-FU doseintensity (percentage of planned 5-FU doses actually received) in thetwo groups was similar (92% vs. 84%) during the treatment course. As ofthe date of date cut-off, 1 patient in the 5-FU/LV/placebo group and 7in the 5-FU/LV/bevacizumab group remained on the assigned initialtherapy. Subsequent therapies, which may have influenced survival, wereused in approximately 50% of patients in both groups, although morepatients in the 5-FU/LV/placebo group were treated with the activeagents irinotecan and oxaliplatin.

Efficacy

Overall survival, the primary endpoint, was longer in the5-FU/LV/bevacizumab group (median, 16.6 months) than in the5-FU/LV/placebo group (median, 12.9 months), demonstrating a trendtoward significance. The hazard ratio of death was estimated to be 0.79(95% CI, 0.56 to 1.10; P=0.16; Table 5 and FIG. 4). The addition ofbevacizumab to 5-FU/LV was associated with increases in medianprogression-free survival (9.2 vs. 5.5 months; hazard ratio=0.50; 95%CI, 0.34 to 0.73; P=0.0002, Table 5 and FIG. 4), response rate (26.0%vs. 15.2%, P=0.055), and median duration of response (9.2 months vs. 6.8months; hazard ratio=0.42; 95% CI, 0.15 to 1.17; P=0.088). A furtheranalysis of treatment effect on overall survival by baselinecharacteristics showed that patients with low serum albumin 3.5 g/dL) atbaseline appeared to derive a significant survival benefit (hazardratio=0.46; 95% CI, 0.29 to 0.74; P=0.001).

TABLE 5 Summary of Efficacy Analysis 5-FU/LV/ 5-FU/LV/ PlaceboBevacizumab Efficacy Parameter (N = 105) (N = 104) P-value Mediansurvival (months) 12.9 16.6 Hazard ratio 0.79 0.160 95% CI 0.56 to 1.10Progression-free survival (months) 5.5 9.2 Hazard ratio 0.50 0.0002 95%CI 0.34 to 0.73 Overall response rate (%) 15.2 26.0 0.055 Completeresponse 0 0 Partial response 15.2 26.0 Duration of response (months)6.8 9.2 Hazard ratio 0.42 0.088 95% CI 0.15 to 1.17 5-FU/LV = 5fluorouracil/leucovorin

Bevacizumab treatment had no detrimental effect on quality of life, andthe TDQ results suggest a possible beneficial effect. The median TDQ asmeasured by the CCS score was 3.0 months in the 5-FU/LV/placebo groupand 3.1 months in the 5-FU/LV/bevacizumab group (hazard ratio=0.79,P=0.188). The median TDQ for placebo-treated and bevacizumab-treatedpatients as measured by secondary TDQ measures was 2.3 and 3.2 months(TOI-C; hazard ratio=0.71, P=0.048) and 2.6 and 3.6 months (totalFACT-C; hazard ratio=0.66, P=0.016).

Safety

A total of 204 patients (104 5-FU/LV/placebo and 1005-FU/LV/bevacizumab) who received at least one dose of study drugcomprised the safety population. A 16% increase (71% versus 87%) intotal grade 3 and 4 toxicities was observed for patients receivingbevacizumab. Adverse events leading to death or study discontinuationwere similar in the two groups, as were adverse events known to beassociated with 5-FU/LV (specifically, diarrhea and leukopenia). Twopatients, both in the 5-FU/LV/bevacizumab group, experienced a bowelperforation event. These events occurred at day 110 and day 338 oftreatment, and both were determined to be associated with a colonicdiverticulum at surgical exploration. One patient died as a result ofthis complication. Previous clinical trials had suggested hemorrhage,thromboembolism, proteinuria, and hypertension as possiblebevacizumab-associated toxicities; however, in this study, no increaseswere seen in venous thrombosis, grade 3 bleeding, or clinicallysignificant grade 3) proteinuria. Arterial thrombotic events (myocardialinfarction, stroke, or peripheral arterial thrombotic event) occurred in10 patients in the 5-FU/LV/bevacizumab group, compared to 5 patients inthe 5-FU/LV/placebo group.

The 5-FU/LV/placebo group had a higher 60-day all-cause mortalitycompared to the 5-FU/LV/bevacizumab group (13.5% vs. 5.0%). Death due todisease progression in the first 60 days was similar (5.8% vs. 4.0%) inthe two groups. In the 5-FU/LV/placebo group, deaths within the first 60days not due to disease progression were attributed to the following:heart failure (1), sepsis (3), diarrhea (2), respiratory failure (1),and pulmonary embolus (1). In the 5-FU/LV/bevacizumab group, the singleearly death not due to disease progression was attributed to amyocardial infarction.

The results of this clinical trial further demonstrate that bevacizumab,a humanized monoclonal antibody against VEGF, provides importantclinical benefit when added to first-line chemotherapy for the treatmentof metastatic colorectal cancer. When compared with 5-FU/LV alone, theaddition of bevacizumab prolonged median survival by 3.7 months,progression-free survival by 3.7 months, and response duration by 2.4months, and increased the response rate by 11%.

These results should be viewed in the context of the study population.Specifically selected were patients who were poor candidates forfirst-line irinotecan-containing therapy, either because of a lowlikelihood of benefit or a high likelihood of treatment-associatedtoxicities. A careful analysis of the pivotal irinotecan trials showedthat clinical benefit from this agent was confined to patients with anormal ECOG performance status (PS=0).21, 22 Advanced age, prior pelvicradiation therapy, impaired performance status, and low serum albuminhave all been reported to increase irinotecan-associated toxicities.23-27 Patients with these characteristics are in need of alternativetherapeutic options. A retrospective subset analysis from a smallerrandomized phase II trial was previously conducted evaluatingbevacizumab and 5-FU/LV in CRC and noted bevacizumab provided asubstantial treatment effect in the subset of patients with baseline PS1 or 2 (median survival, 6.3 months vs. 15.2 months), in the subset aged≧65 years (11.2 months vs. 17.7 months), and in the subset with serumalbumin<3.5 (8.1 months versus 14.1 months). These results encouraged usto design the current trial, specifically including a poor-prognosisstudy population and powering the trial to detect a large treatmenteffect on survival. We were largely successful in enrolling a populationdifferent from that in the concurrently conducted pivotal trial ofIFL/placebo versus IFL/bevacizumab. Compared with the pivotal trial,patients in the present trial had a higher median age (72 vs. 61 years)and substantially more patients had a performance status>0 (72% vs. 43%)and albumin≦3.5 mg/dL (46% vs. 33%).

Despite this high-risk study population, the regimen of5-FU/LV/bevacizumab appeared to be well tolerated. The well-describedbevacizumab-associated adverse event of grade 3 hypertension was seen in16% of the 5-FU/LV/bevacizumab group versus 3% in the 5-FU/LV/placebogroup. No cases of grade 4 hypertension occurred. Proteinuria of anygrade was seen in 38% of the 5-FU/LV/bevacizumab group versus 19% of the5-FU/LV/placeb group; however, only a single patient in the bevacizumabgroup developed grade 3 proteinuria, and there were no cases of grade 4proteinuria. No increases in grade 3 or 4 bleeding or venous thromboticevents were seen in bevacizumab-treated patients. There was an imbalancein the incidence of arterial thrombotic events: 10% in the5-FU/LV/bevacizumab group compared with 4.8% in the 5-FU-/LV placebogroup. A similar imbalance was noted in the pivotal bevacizumab trial(1.0% in the IFL/placebo group and 3.3% in the IFL/bevacizumab group).The more advanced age of the population included in the present studymay have contributed to a higher overall incidence of this adverseevent, however the imbalance in both studies is noteworthy. Large,observational safety trials may be required to further define theincidence and potential risk factors for these, and other, uncommonadverse events associated with bevacizumab therapy.

In summary, these data demonstrate that bevacizumab, when combined withbolus 5-FU/LV, provides substantial clinical benefit for patients withpreviously untreated metastatic colorectal cancer who are deemed to bepoor candidates for irinotecan-containing therapy. Together with thepivotal trial results, these data strengthen the evidence thatbevacizumab-based, 5-FU/LV-containing therapy should be considered astandard option for the initial treatment of metastatic colorectalcancer.

1-46. (canceled)
 47. A method of treating cancer in a patient comprisingadministering to the patient an effective amount of bevacizumab, whereinthe patient has a grade III hypertensive event resulting from thebevacizumab administration, the method further comprising administeringto the patient an antihypertensive agent in an amount sufficient tomanage the grade III hypertensive event while continuing to treat thepatient with bevacizumab, the continued bevacizumab treatment beingcarried out without altering the dosage regimen.
 48. The method of claim47, wherein the method further comprises administering one or morechemotherapeutic agents.
 49. The method of claim 48, wherein the one ormore chemotherapeutic agents is one or more of an alkylating agent,antimetabolite, pyrimidine analog, vinca alkyloid, epipodophyllotoxin,antibiotic, topoisomerase inhibitor, interferon, platinum coordinationcomplex, or taxoid.
 50. The method of claim 49, wherein the one or morechemotherapeutic agents is one or more of a pyrimidine analog,5-fluorouracil (5-FU), leucovorin, irinotecan, paclitaxel, oxaliplatin,carboplatin, cisplatin, doxorubicin, topotecan, or interferon-alpha. 51.The method of claim 50, wherein the one or more chemotherapeutic agentsis one or more of 5-FU, leucovorin, irinotecan, or oxaliplatin.
 52. Themethod of claim 51, wherein the one or more chemotherapeutic agents is5-FU and leucovorin; 5-FU, leucovorin, and irinotecan; or 5-FU,leucovorin, and oxaliplatin.
 53. The method of claim 48, wherein thecancer is colorectal cancer, rectal cancer, glioblastoma, non-small celllung cancer, cervical cancer, peritoneum cancer, renal cancer, ovariancancer, or mesothelioma.
 54. The method of claim 51, wherein the canceris metastatic colorectal cancer.
 55. The method of claim 52, wherein thecancer is metastatic colorectal cancer.
 56. The method of claim 50,wherein the cancer is metastatic non-small cell lung cancer.
 57. Themethod of claim 50, wherein the cancer is metastatic cervical cancer.58. The method of claim 50, wherein the cancer is metastatic ovariancancer or metastatic peritoneum cancer.
 59. The method of claim 50,wherein the cancer is metastatic renal cancer.
 60. The method of claim47, wherein the cancer is glioblastoma.
 61. The method of claim 47,wherein the bevacizumab is administered to the patient bi-weekly atabout 10 mg/kg.
 62. The method of claim 50, wherein the bevacizumab isadministered to the patient at about 15 mg/kg every 3 weeks.
 63. Themethod of claim 50, wherein the antihypertensive agent is acalcium-channel blocker.
 64. The method of claim 50, wherein theantihypertensive agent is an angiotensin-converting-enzyme inhibitor.65. The method of claim 50, wherein the antihypertensive agent is adiuretic.
 66. The method of claim 50, wherein the patient does not haveclinically significant cardiovascular disease prior to the bevacizumabadministration.
 67. The method of claim 54, wherein one of the one ormore chemotherapeutic agents is 5-FU.